Jacques Van Snick

Mast cells are essential intermediaries in regulatory T-cell tolerance

Contrary to the proinflammatory role of mast cells in allergic disorders, the results obtained in this study establish that mast cells are essential in CD4+CD25+Foxp3+ regulatory T (TReg)-cell-dependent peripheral tolerance. Here we confirm that tolerant allografts, which are sustained owing to the immunosuppressive effects of TReg cells, acquire a unique genetic signature dominated by the expression of mast-cell-gene products. We also show that mast cells are crucial for allograft tolerance, through the inability to induce tolerance in mast-cell-deficient mice. High levels of interleukin (IL)-9—a mast cell growth and activation factor—are produced by activated TReg cells, and IL-9 production seems important in mast cell recruitment to, and activation in, tolerant tissue. Our data indicate that IL-9 represents the functional link through which activated TReg cells recruit and activate mast cells to mediate regional immune suppression, because neutralization of IL-9 greatly accelerates allograft rejection in tolerant mice. Finally, immunohistochemical analysis clearly demonstrates the existence of this novel TReg–IL-9–mast cell relationship within tolerant allografts.

Loss of SOCS3 expression in T cells reveals a regulatory role for interleukin-17 in atherosclerosis

Atherosclerosis is an inflammatory vascular disease responsible for the first cause of mortality worldwide. Recent studies have clearly highlighted the critical role of the immunoinflammatory balance in the modulation of disease development and progression. However, the immunoregulatory pathways that control atherosclerosis remain largely unknown. We show that loss of suppressor of cytokine signaling (SOCS) 3 in T cells increases both interleukin (IL)-17 and IL-10 production, induces an antiinflammatory macrophage phenotype, and leads to unexpected IL-17–dependent reduction in lesion development and vascular inflammation. In vivo administration of IL-17 reduces endothelial vascular cell adhesion molecule–1 expression and vascular T cell infiltration, and significantly limits atherosclerotic lesion development. In contrast, overexpression of SOCS3 in T cells reduces IL-17 and accelerates atherosclerosis. We also show that in human lesions, increased levels of signal transducer and activator of transcription (STAT) 3 phosphorylation and IL-17 are associated with a stable plaque phenotype. These results identify novel SOCS3-controlled IL-17 regulatory pathways in atherosclerosis and may have important implications for the understanding of the increased susceptibility to vascular inflammation in patients with dominant-negative STAT3 mutations and defective Th17 cell differentiation.

IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells

The development of T helper (TH)17 and regulatory T (Treg) cells is reciprocally regulated by cytokines. Transforming growth factor (TGF)-β alone induces FoxP3+ Treg cells, but together with IL-6 or IL-21 induces TH17 cells. Here we demonstrate that IL-9 is a key molecule that affects differentiation of TH17 cells and Treg function. IL-9 predominantly produced by TH17 cells, synergizes with TGF-β1 to differentiate naïve CD4+ T cells into TH17 cells, while IL-9 secretion by TH17 cells is regulated by IL-23. Interestingly, IL-9 enhances the suppressive functions of FoxP3+ CD4+ Treg cells in vitro, and absence of IL-9 signaling weakens the suppressive activity of nTregs in vivo, leading to an increase in effector cells and worsening of experimental autoimmune encephalomyelitis. The mechanism of IL-9 effects on TH17 and Tregs is through activation of STAT3 and STAT5 signaling. Our findings highlight a role of IL-9 as a regulator of pathogenic versus protective mechanisms of immune responses.

Development of an anti-IL-17A auto-vaccine that prevents experimental auto-immune encephalomyelitis

IL‐17 has been associated with multiple inflammatory disorders such as rheumatoid arthritis, asthma and multiple sclerosis. As these diseases require long‐term treatment we turned to an auto‐vaccine strategy for IL‐17 neutralization in vivo. Mouse IL‐17A was covalently linked to ovalbumin and used to immunize C57BL/6 mice. This vaccine induced the production of antibodies that blocked IL‐17A bioactivity in vitro but did not react with the other IL‐17 isoforms, including IL‐17F. As the half‐life of the Ab titers after the last immunogen administration was approximately 4u2004months, the vaccine provides for long lasting and selective inhibition of IL‐17A activity in vivo. A monoclonal Ab (mAb) derived from these mice showed the same specificity for IL‐17A. To test the ability of the vaccine to confer protection against an IL‐17‐dependent disorder, SJL mice were vaccinated with IL‐17‐OVA and encephalomyelitis (EAE) was induced by proteolipid protein (PLP) peptide 139–151. Vaccinated mice were completely protected against the disease. The above‐mentioned anti‐IL‐17A mAb also prevented EAE development. The absence of clinical symptoms contrasted with unaltered PLP‐induced cytokine production in vitro and unmodified anti‐PLP IgG titers and isotypes. These results suggest that an anti‐IL‐17A auto‐vaccine offers new perspectives for therapy of autoimmune diseases.

IL-9 Is a Susceptibility Factor in Leishmania major Infection by Promoting Detrimental Th2/Type 2 Responses

IL-9 is a cytokine produced by Th2 cells, induced during Leishmania major infection. Because the role of IL-9 in leishmaniasis is currently unknown, IL-9-deficient mice were generated by immunization with mouse IL-9 coupled to OVA. This produced strong and long-lasting neutralizing anti-IL-9 Abs in vivo. Anti-IL-9 vaccination showed protective effects, because it enabled L. major-infected nonhealer BALB/c mice to better resist to leishmaniasis with doubling the time span until pathological disease progression occurred. Increased resistance was also demonstrated by moderate footpad swelling and histopathology due to reduced parasite burden compared with sham-immunized BALB/c mice. Mechanistically, IL-9 neutralization in BALB/c mice resulted in a reduction of detrimental Th2/type 2 responses with an observed shift toward protective Th1 immune responses. This led to an alteration from alternative to classical macrophage activation with subsequent enhanced killing effector functions, as demonstrated by increased NO production but reduced arginase 1-mediated macrophage responses. Conclusively, the data show that IL-9 is a susceptible factor in leishmaniasis. They further suggest that IL-9 is able to influence Th dichotomy in leishmaniasis by promoting detrimental Th2/type 2 responses in BALB/c mice. The results extend efforts made to generate autoantibodies capable of regulating biological processes, with IL-9 a potential drug target against leishmaniasis.

Development of an anti-IL-12 p40 auto-vaccine: protection in experimental autoimmune encephalomyelitis at the expense of increased sensitivity to infection

IL‐12 and IL‐23, which share the IL‐12 p40 subunit, have been ascribed central roles in many autoimmune disorders. We describe here an anti‐IL‐12 (αIL‐12) auto‐vaccine that potentially blocks both factors in vivo. Immunization of mice with mouse IL‐12 coupled to OVA or Pan DR epitope (PADRE) peptide induced Ab directed against the IL‐12 p40 subunit, which prevented IFN‐γ production in response to IL‐12 administration in vivo. Experimental autoimmune encephalomyelitis, an IL‐23‐dependent disease model, induced in SJL mice with a proteolipid protein (PLP) peptide was almost undetectable after αIL‐12 vaccination. Myelin oligodendrocyte glycoprotein (MOG)‐induced disease in C57BL/6 mice was also significantly inhibited. This protection correlated with inhibited Th1 cytokine responses in vitro and with an increase in the IgG1/IgG2a anti‐PLP Ab balance. Detrimental consequences of αIL‐12 vaccination were evaluated in C57BL/6 mice infected with Leishmania major (L.m.). While delayed‐type hypersensitivity (DTH) suppression and immunoglobulin as well as interleukin production patterns reflected a major shift toward a Th2‐type response, L.m. growth was still significantly retarded as compared to that seen in susceptible BALB/c mice. However, vaccinated animals ultimately failed to control parasite expansion. These results suggest that some chronic autoimmune diseases may benefit from αIL‐12 vaccination at the expense of reduced, but not completely abrogated, cell‐mediated immunity.

Signals through 4-1BB inhibit T regulatory cells by blocking IL-9 production enhancing antitumor responses

Previous studies from our laboratory indicate that intratumoral (i.t.) injections of CpG-ODN are the most effective adjuvant strategy to induce an antitumor immune response in tolerant BALB-neuT mice but insufficient for tumor eradication. We evaluated whether this treatment strategy could be enhanced by the presence of anti-OX40 and anti-4-1BB antibodies. Treatment with anti-4-1BB resulted in a greater antitumor response than anti-OX40. The results indicate that anti-4-1BB but not anti-OX40 inhibited the suppressive function of T regulatory cells (Tregs). Through microarray analysis we evaluated the mechanism by which anti-4-1BB inhibits iTregs using the Foxp3-GFP mice. We observed specific transcriptional differences in over 100 genes in iTregs treated with anti-4-1BB, and selected those genes that remained unaffected by exposure to anti-OX40. Interleukin 9 was transcriptionally down-regulated 28-fold by anti-4-1BB treatment, and this was matched by a significant reduction of IL-9 secretion by iTregs. Furthermore, blockade of the common γ-chain receptor resulted in the inhibition of iTreg-suppressive function. More importantly, neutralization of IL-9 plus i.t. injections of CpG-ODN induces tumor rejection in BALB-neuT and MUC-1 tolerant transgenic mice. These results indicate that IL-9 plays a role in iTreg biology during the tumor inflammatory process enhancing/promoting the suppressive function of these cells and that the blockade of IL-9 could serve as a novel strategy to modulate the function of Tregs to enhance the antitumor effect of tumor vaccines.

TGF-β interactions with IL-1 family members trigger IL-4-independent IL-9 production by mouse CD4(+) T cells.

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4+ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4+ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.

Anti-IL-17A autovaccination prevents clinical and histological manifestations of experimental autoimmune encephalomyelitis.

Abstract:u2002 Excessive or inappropriate production of IL‐17A has been reported in diseases such as rheumatoid arthritis, asthma, and multiple sclerosis. The potential clinical relevance of these correlations was suggested by the protective effects of anti‐IL‐17A monoclonal antibodies in various mouse disease models. However, the chronic nature of the corresponding human afflictions raises great challenges for Ab‐based therapies. An alternative to passive Ab therapy is autovaccination. Covalent association of self‐cytokines with foreign proteins has been reported to induce the production of antibodies capable of neutralizing the biological activity of the target cytokine. We recently reported that cross‐linking of IL‐17A to ovalbumin produced highly immunogenic complexes that induced long‐lasting IL‐17A‐neutralizing antibodies. Vaccinated SJL mice were completely protected against experimental autoimmune encephalomyelitis (EAE) induced by proteolipid protein peptide (PLP 139–151), and a monoclonal anti‐IL‐17A Ab (MM17F3), derived from C57Bl/6 mice vaccinated against IL‐17A‐OVA, also prevented disease development. Here we report that this Ab also protects C57Bl/6 mice from myelin oligdendrocyte glycoprotein (MOG)‐induced EAE. Histological analysis of brain sections of C57Bl/6 mice treated with MM17F3 showed a complete absence of inflammatory infiltrates and evidence for a marked inhibition of chemokine and cytokine messages in the spinal cord. These results further extend the analytical and therapeutic potential of the autovaccine procedure.

PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract

Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab)2 and anti-IL-13 Fab greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4h post-delivery, whereas the clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab)2 and Fab contents in the lungs had decreased to 10 and 14% of the dose initially deposited, respectively. However, this value was 40% for both PEG40-F(ab)2 and PEG40-Fab. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab)2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways.